Global Analysis of Protein Lysine Succinylation Profiles and Their

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Global analysis of protein lysine succinylation profiles and their overlap with lysine acetylation in the marine bacterium Vibrio parahaemolyticus Jianyi Pan, Ran Chen, Chuchu Li, Weiyan Li, and Zhicang Ye J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00485 • Publication Date (Web): 15 Sep 2015 Downloaded from http://pubs.acs.org on September 16, 2015

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Journal of Proteome Research

Global analysis of protein lysine succinylation profiles and their overlap with lysine acetylation in the marine bacterium Vibrio parahaemolyticus

Jianyi Pan*, Ran Chen, Chuchu Li, Weiyan Li and Zhicang Ye

Institute of Proteomics and Molecular Enzymology, School of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China

*Corresponding author: Dr. Jianyi Pan, School of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, 310018, China. E-mail: [email protected], Tel: +86 571 86843748, Fax: +86 571 86843745.

1

ACS Paragon Plus Environment


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ABSTRACT Protein lysine acylation, including acetylation and succinylation, has been found to be a major post-translational modification (PTM) and is associated with the regulation of cellular processes that are widespread in bacteria. Vibrio parahaemolyticus is a model marine bacterium that causes seafood-borne illness in humans worldwide. The lysine acetylation of V. parahaemolyticus has been extensively characterized in our previous work, and here, we report the first global analysis of lysine succinylation and the overlap between the two types of acylation in this bacterium. Using high accuracy nano LC-MS/MS combined with affinity purification, we identified 1931 lysine succinylated peptides matched on 642 proteins, with the quantity of the succinyl-proteins accounting for 13.3% of the total proteins in cells. Bioinformatics analysis results showed that these succinylated proteins are involved in almost every cellular process, particularly in protein biosynthesis and metabolism, and are distributed in diverse subcellular compartments. Moreover, several sequence motifs were identified, including succinyl-lysine flanked by a lysine or arginine residue at the -8, -7 or +7 position and without these residues at the -1 or +2 position, and these motifs differ from those found in other bacteria and eukaryotic cells. Furthermore, a total of 517 succinyl-lysine sites (26.7%) on 288 proteins (44.9%) were also found to be acetylated, suggesting extensive overlap between succinylation and acetylation in this bacterium. This systematic analysis provides a promising starting point for further investigations of the physiologic and pathogenic roles of lysine succinylation and acetylation in V. parahaemolyticus. KEYWORDS:

lysine

succinylation,

lysine

acetylation,

post-translational modification (PTM), V. parahaemolyticus

2


sequence

motif,

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INTRODUCTION Protein post-translational modifications (PTMs) are dynamic and reversible modifications of proteins that naturally occur both in eukaryotic and prokaryotic cells. PTMs are known as one of the most efficient strategies for expanding the functional diversity of existing proteins and for increasing the control of cellular functions.1,2 Among the 20 amino acid residues in proteins, lysine is a frequent target for modification,3 and a plethora of lysine PTMs, such as methylation,4 ubiquitination,5 SUMOylation,6 acetylation,7,8 succinylation,9 crotonylation,10 malonylation,11 propionylation and butyrylation,12,13 have been identified at large scales in prokaryotic and/or eukaryotic cells in the last few years. Of these modifications, acylation, especially acetylation, at lysine residues has been extensively studied recently in the proteomes of diverse organisms, and it is accepted as an important regulatory mechanism for almost every aspect of cellular physiology and pathology. Lysine succinylation, one type of lysine acylation similar to lysine acetylation, is a newly discovered PTM. This type of modification was found to exist in both eukaryotes and prokaryotes just a few years ago, and proteomic screenings identified succinyl-proteins more recently. To date, lysine succinylation has been profiled both in eukaryotes, including HeLa cells, yeast and mouse liver mitochondria,14-16 and protozoan parasite Toxoplasma gondii,3 and in bacteria, including Escherichia coli15,17 and Mycobacterium tuberculosis.18,19 Based on the obtained proteomic lysine succinylation datasets, this PTM is evolutionarily conserved in various organisms and occurs extensively in proteins associated with diverse cellular processes, such as metabolism and translation. Moreover, in comparison with lysine acetylation, lysine succinylation responds more dynamically to changes in growth conditions or genetic mutations.17 Thus, lysine succinylation is likely to have a stronger role in cellular 3



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regulation than lysine acetylation.20 Therefore, comprehensive identification of lysine succinylation may be more critical to understanding the regulatory mechanism of cells, which will in turn facilitate disease treatment and prevention. Vibrio parahaemolyticus is a Gram-negative marine bacterium, a typical pathogenic member of the Vibrio genus in the Vibrionaceae family.21 The bacterium is found worldwide in marine, estuarine and coastal environments. It is a leading global cause of bacterial seafood-borne illness, such as acute gastroenteritis and diarrhea, which is associated with the consumption of contaminated raw or undercooked seafood. In rare cases, V. parahaemolyticus causes septicemia due to bacterial exposure in open wounds, and in immunocompromised individuals, this septicemia correlates with a high mortality rate.22,23 Currently, it is known that V. parahaemolyticus are multi-serotype bacteria, containing at least 12 different O antigens and 70 different K antigens in their capsule.24 A typical clinical strain, possessing serotype O3:K6, has been found to be extremely virulent and pathogenic to humans and is considered to be one of the major agents of seafood-borne diseases.25 It is also the root cause of an increasing number of worldwide outbreaks of gastroenteritis.26 Although the mechanisms of universal virulence control have not been clarified thoroughly in the species to date, it is interesting that protein lysine acetylation27 and AMPylation28 have been revealed recently as being associated with bacterial virulence. These findings suggest that protein PTMs may be key mechanisms for virulence or pathogenicity in V. parahaemolyticus. Therefore, it is necessary to conduct systematic studies of PTMs proteome-wide to clearly reveal the regulatory roles both in physiological processes and in the pathogenicity of V. parahaemolyticus. Fortunately, the entire genome of V. parahaemolyticus serotype O3:K6 (strain 4


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RIMD 2260133) has been sequenced by Makino et al.,29 and this enables us to perform a global analysis of diverse PTMs in this species. Based on this remarkable work, we have initiated an intensive investigation of protein lysine acetylation in V. parahaemolyticus and previously obtained important findings.30 In the present study, we present the first systematic analysis of protein lysine succinylation identified using nano LC-MS/MS in combination with affinity purification and the overlap between succinylation and acetylation in this bacterium. In total, 1931 lysine succinylated sites on 642 proteins were identified in diverse biological processes and cellular localizations. Among these identified sites and proteins, succinylation at 517 lysine sites (26.7%) on 288 proteins (44.9%) was found overlapping with acetylation. Moreover, several sequence motifs with succinyl-lysine flanked by a lysine or arginine at the -8, -7 or +7 position and without the two residues at -1 or +2 position, differing from the motifs found in other bacteria and eukaryotic cells, were also identified. Overall, our findings provide a promising platform for the further investigation of lysine succinylation and acetylation in physiological and pathogenic processes in V. parahaemolyticus.

MATERIALS AND METHODS Bacterial Strains and Growth Conditions Vibrio parahaemolyticus serotype O3:K6 (strain RIMD 2210633) was grown overnight in MLB medium (Luria-Bertani medium containing 3% NaCl) at 30 °C. The seed culture was inoculated in fresh MLB medium at a ratio of 1:100 and then incubated at 30 °C with shaking. The bacterial cells were collected at an OD600 of 0.8 by centrifugation at 6000 g and 4 °C for 5 min and then washed three times with sterile PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). 5



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Preparation of Proteins and In-solution Digestion The preparation of proteins and in-solution trypsin digestion were carried out as described in our previous work.30 Briefly, the bacterial pellets were resuspended in lysis buffer (8 M urea, 2 mM EDTA, 5 mM DTT, and 1% (v/v) protease inhibitor cocktail Set III (Calbiochem)) and then sonicated on ice. The resulting samples were centrifuged at 4°C and 20,000 g for 10 min to remove unbroken cells and debris. Then, the protein content in the supernatant was determined using a 2-D Quant kit (GE Healthcare). The proteins were precipitated with 20% trichloroacetic acid (TCA) overnight at 4°C, and the resulting precipitate was washed three times with ice-cold acetone. Then, the air-dried precipitate was resuspended in 100 mM NH4HCO3 and digested with trypsin (Promega) at an enzyme/substrate ratio of 1:50 for 15 h at 37°C. The resulting peptides were reduced with 5 mM DTT at 56°C for 45 min and subsequently alkylated with 15 mM iodoacetamide (IAA) for 30 min at room temperature (RT) in the dark. To terminate the reaction, 30 mM cysteine was added and incubated for 20 min at RT. Then, to ensure complete digestion, trypsin was added at an enzyme/substrate ratio of 1:100 and incubated for 4 h. The tryptic peptides were dried in a SpeedVac.

Immunoaffinity Enrichment of Lysine Succinylated Peptides Lysine succinylated peptides were enriched by the immunoaffinity procedure as previously described.17 In brief, the dried digests were redissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, pH 8.0 and 0.5% Nonidet P-40) and incubated with anti-succinyllysine agarose beads (PTM Biolabs) at a ratio of 20 µl of beads per milligram of proteins at 4°C overnight with gentle rotation. After incubation, 6


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the supernatant was removed and the beads were carefully washed three times with NETN buffer, twice with NET buffer (100 mM NaCl and 1 mM EDTA, 50 mM Tris-Cl, pH 8.0), and once with water. The bound peptides were eluted from the beads with 1% trifluoroacetic acid (TFA) and dried in a SpeedVac. The obtained peptides were desalted with C18 Zip Tips (Millipore) according to the manufacturer’s instructions and then subjected to HPLC-MS/MS analysis.

Nano HPLC-MS/MS Analysis The dried peptides were dissolved in 3 µl of HPLC buffer A (2% Acetonitrile (ACN) and 0.1% formic acid (FA) in water) and then analyzed by online nano LC-MS/MS using an easy nLC1000 nano-UPLC system (Thermo Scientific) coupled to a Q ExactiveTM Plus hybrid quadrupole-OrbitrapTM mass spectrometer (Thermo Scientific). The peptide was eluted onto an Acclaim PepMap RSLC C18 capillary column (50 µm × 15 cm, Dionex) at a flow rate of 300 nl/min with a 34 min gradient from 5% to 40% HPLC buffer B (80% ACN and 0.1% FA in water), followed by 2 min of 40% buffer B, then 2 min from 40% to 80% buffer B, and finally 4 min of 80% buffer B. The eluted peptides were then ionized and sprayed into the mass spectrometer by a nanospray-ionization (NSI) source. Intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were selected for MS/MS using 27% NCE step to 30% NCE; ion fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions above a threshold ion count of 2E4 in the MS survey scan with 15.0s dynamic exclusion. The electrospray voltage applied was 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the ion trap; 5E4 ions were accumulated for generation of MS/MS 7



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spectra. For MS scans, the m/z scan range was 350 to 1800.

Database Searching All of the raw data files obtained from the mass spectrometry analysis were processed using MaxQuant software (version 1.4.1.2). The mass spectra were compared against the protein database of V. parahaemolyticus serotype O3:K6 (strains RIMD 2210633) (containing 4,823 sequences) from UniProt (http://www.uniprot.org/uniprot). Trypsin/P was specified as a cleavage enzyme, and the search allowed up to 3 missing cleavages, 5 charges and 5 modifications per peptide. Mass error was set to 20 ppm for first search, 5 ppm for main search and 0.02 Da for fragment ions. The mass error was set to 5 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethylation on Cys was specified as a fixed modification, and oxidation on Met, succinylation on lysine (lysine +100.01604) and acetylation on protein N-terminal were specified as variable modifications. The false discovery rate (FDR) thresholds for proteins, peptides, and modification sites were specified as 1%. Minimum peptide length was set at 7. The site localization probability was set as > 0.75. The cut-off score for peptides was set to 40.

Bioinformatics Analysis of Lysine Succinylated Peptides and Proteins The bioinformatics analysis of the identified lysine succinylated peptides and proteins was described in detail previously.30 Briefly, Gene Ontology (GO) annotation was performed from the UniProt-GOA database (www. http://www.ebi.ac.uk/GOA/). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was annotated using online service tool KEGG Automatic Annotation Server (KAAS), and the annotation result was mapped using the online service tool KEGG Mapper. Fisher’s 8


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exact test (two-tailed test) was used to test for GO\KEGG pathway\Pfam domain enrichment analysis. Amino acid sequence motifs (ten amino acids upstream and downstream of the succinylated lysine) were analyzed using motif-X. Motif-based clustering analyses were also performed and cluster membership was visualized using a heat map. Overlap between succinylated peptides/proteins and our previously published acetylated peptides/proteins was presented.

RESULTS AND DISCUSSION Detection of Lysine-succinylated Peptides and Proteins in V. parahaemolyticus Lysine succinylation is a newly identified protein PTM that occurs both in eukaryotic and prokaryotic cells.9 Proteome-wide lysine succinylation of several bacteria. including E. coli15,17 and M. tuberculosis,18,19 was also recently identified. This type of PTM has not been found in the proteins of V. parahaemolyticus, a typical marine bacterium, and the lysine succinylome (all lysine succinylated proteins in a cell) has not been reported. In the present study, a proteomic method combining affinity enrichment by the succinyl-lysine antibody with highly sensitive LC-MS was used to identify the global lysine succinylated proteins in V. parahaemolyticus. The obtained MS raw data were compared against the UniProt V. parahaemolyticus serotype O3:K6 protein database (altogether 4,823 protein sequences). A total of 1931 succinylated-lysine sites (in 1931 peptides) from 642 proteins were identified with high confidence, with an average peptide score of 77.7 and an average mass error of 0.05. Detailed information for all of the identified lysine succinylation peptides and the matched proteins are presented in Table S1 in Supporting Information, and the representative MS/MS spectra of six lysine succinyl-peptides of protein SucC (succinyl-CoA ligase, ADP-forming, subunit beta), GKsucAGGVELHDTK, 9



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AGGVELHDTKsucEGVK, EGVKsucEFAQK, EFAQKsucWLGK, VAEETPELIHKsucAAIDPLVGPQAYQGR and LGLEGDQIKsucQFVK with succinylated sites at K56, K66, K70, K75, K146 and K175, respectively, are shown in Figure S1 in Supporting Information. All of the identified lysine succinylated peptides vary in length from 7-35 amino acids, and 11% of which contain 9 amino acids and the total number of peptides with lengths of 8, 10, 11 and 12 amino acids account for 9% (Figure 1A and Table S1 in Supporting Information). Among the 642 total succinylated proteins, 40% only have one lysine succinylated site, and proteins with 2, 3, 4 and 5 succinylated sites account for 20%, 12%, 7% and 5% of the total, respectively (Figure 1B and Table S1 in Supporting Information). In addition, 4% of the proteins are modified at 10 or more lysine sites (Figure 1B). The lysine site distribution is very similar to that found in prokaryotes (E. coli)17 and eukaryotes (T. gondii)3 and showed a minor variation compared to that of M. tuberculosis in which 51% of proteins possessed only one site.18 Notably, five proteins were found to possess more than 20 succinylated sites, and these proteins are: 30S ribosomal protein S1 (22 sites); DNA-directed RNA polymerase subunit β’ (21 sites); DNA-directed RNA polymerase subunit β (20 sites); pyruvate dehydrogenase E1 component (20 sites); and formate acetyltransferase (20 sites) (Table S1 in Supporting Information). Interestingly, the 30S ribosomal protein S1 was also identified as a heavily succinylated protein in E. coli.17 In addition, two subunits (β’ and β) of RNA polymerase are extensively modified, indicating that this PTM may be very important for transcription in the bacterium. All 642 identified succinyl-proteins account for 13.3% of the total proteins in V. parahaemolyticus. Both the number of succinyl-proteins and the percentage of succinyl-proteins to total proteins are similar to other identified Gram-negative 10


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bacterial species, including E. coli K-12 substrain DH10B,17 M. tuberculosis H37Rv19 and M. tuberculosis XDR18 (Table 1). Notably, although the number of lysine succinylated peptides between E. coli substrain DH10B and E. coli BW25113 are similar, the number of matched succinyl-proteins varies markedly, as 670 and 990 different proteins were matched from 2580 and 2572 peptides in these two strains, respectively (Table 1), suggesting a significant difference in the distribution of lysine-succinylated sites in their proteins.

Functional Annotation and Subcellular Localization of Lysine Succinylated Proteins Protein lysine succinylation has been conjectured to be important for regulating cellular functions.20 To elucidate the potential roles of lysine succinylation in V. parahaemolyticus, all of the identified succinyl-proteins were submitted to Gene Ontology (GO) functional classification analysis based on their biological process, molecular function and subcellular localization (Figure 2 and Table S2 in Supporting Information). In the classification of biological processes, two major classes of succinyl-protein are associated with metabolism and cellular process, accounting for 40% and 38% of the total succinyl-proteins, respectively (Figures 2A). The percentage of each of these two classes of succinyl-proteins to the total number of succinyl-proteins was consistent with that of M. tuberculosis,18 at 44% and 38 %, respectively. These findings suggest the essential role of protein lysine succinylation is in metabolism and cellular processes in bacterial cells. In addition, there are 1% succinyl-proteins related to signaling, a percentage of this class much higher than that found in M. tuberculosis H37Rv, where the percentage was 0.12%.19 The classification result for molecular function showed that the most succinylated 11



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proteins were related to catalytic activity, and the percentage of proteins in this class was 45% (Figure 2B). Another large succinyl-protein class was binding proteins, which accounted for 40% of all of the succinylated proteins. These results are in agreement with the classification results of the biological process. Moreover, the classification results for molecular function were also similar to previous reports that showed that the percentage of the classes of catalytic activity and binding were 50.8% and 45.5%, respectively, in M. tuberculosis H37Rv,19 and 49% and 42% in M. tuberculosis XDR.18 The analysis of the GO functional classification of the succinyl-proteins indicates that a wide ranges of proteins in biological processes and molecular functions can be succinylated in V. parahaemolyticus. The subcellular localization result of all of the identified succinyl-proteins showed that most of the proteins (90%) are distributed in the cytoplasm and that succinyl-proteins located in the periplasmic space, inner membrane, outer membrane and extracellular account for 5%, 3%, 1% and 1% of the total succinyl-proteins, respectively (Figure 2C). The overall localization trend of succinyl-proteins is similar to the previous findings obtained in M. tuberculosis XDR.18

Enrichment Analysis of Lysine Succinylated Proteins To better understand the biological function of succinylated proteins, we further performed an enrichment analysis in the GO, KEGG pathway and Pfam domain (Figures 3 and Table S3 in Supporting Information). As shown in Figure 3A (blue bars), GO enrichment analysis based on the biological process category showed that the processes associated with tRNA aminoacylation for protein translation, translation, tRNA aminoacylation and amino acid activation were found to be significantly enriched. It is interesting that all of the identified enriched processes are related to 12


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translation and protein biosynthesis. Similar results were also found in the GO enrichment analysis

for

the

molecular function

category,

in which

the

succinyl-proteins with rRNA binding, structural constituent of the ribosome, translation factor activity (nucleic acid binding) and aminoacyl-tRNA ligase activity were most likely enriched (Figure 3A, red bars). These proteins are mainly involved in the ribosome and protein biosynthesis. In agreement with this observation, the succinyl-proteins were significantly enriched in the ribosome and ribonucleoprotein complex when enrichment analysis of the cellular components was performed (Figure 3A, green bars). Altogether, the GO enrichment results suggest that proteins related to protein biosynthesis, translation or ribosome have a high tendency to be succinylated in V. parahaemolyticus. Furthermore, the enrichment analysis of the Pfam domain and KEGG pathway both demonstrate that proteins related to protein biosynthesis are prone to succinylation (Figure 3B and C). Protein domains associated with aminoacyl-tRNA synthetase, translation elongation factor, the translation protein and ribosomal protein, and the KEGG pathways of ribosome and aminoacyl-tRNA biosynthesis were significantly enriched, as shown in Figure 3B and Figure 3C, respectively. In addition, besides the enrichment of succinyl-proteins related to protein biosynthesis, proteins associated with central metabolism, including the pathways of glycolysis/gluconeogenesis, pentose phosphate, and citrate cycle, were also found to be significantly enriched (Figure 3C and Figure S2 in Supporting Information)(also see Figure 7B). In combination, the enrichment analyses of GO annotation, Pfam domain and KEGG pathway suggest that lysine succinylation at proteins related to protein biosynthesis and central metabolism is particularly essential to the physiological function of V. parahaemolyticus. Interestingly, similar results have also been found in other 13



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Gram-negative bacteria, specifically M. tuberculosis18,19 and E. coli.17

Motifs of Lysine Succinylated Peptides The data sets comprising the identification of succinylated peptides proteome-wide can aid in the determination of the preferences for specific amino acid residues at particular positions surrounding the succinylated lysine as reported previously.18 Therefore, we utilized the Motif-X extractor web tool, which is software that was designed to extract overrepresented patterns from any set of sequences,31 to determine the sequence motifs from all of the succinylated peptides in V. parahaemolyticus. A total of five conserved motifs, with amino acid sequences from -9 to +9 surrounding the succinylated lysine, were extracted from 671 succinylated peptides (Figure 4 and Table 2). These motifs are R******Ksuc, K*******Ksuc, K******Ksuc, Ksuc******K and Ksuc******R (Ksuc indicates the succinylated lysine, and asterisk (*) indicates a random amino acid residue) (Figure 4A). In accordance with these findings, the analysis of the frequency of amino acids flanking succinylated lysine showed that the lysine (K) at -7 to -9, +6 to +8, particularly at positions -8, -7 and +7, and arginine (R) at -7 and +7 were significantly preferred (Figure 4B). Furthermore, from Figure 4B, we also found that the frequencies of K at -1 and +2 and R at -1 in the motifs were the lowest. These results suggest that succinylation is preferred at lysine residues that are surrounded by an alkaline residue with a long side-chain (K or R) at position of -7, -8 or +7 and without these amino acids at the position of -1 or +2. It is interesting that some of these succinylation motifs are very similar to segments of acetylation motifs, such as Kac****K and Kac****R (Kac indicates the acetylated lysine), which can be extracted from this bacterium as described in our previous work.30 However, all five motifs were unique compared to those found in other bacteria, i.e., EKsuc in M. 14


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tuberculosis;19 EKsuc and K*****Ksuc in M. tuberculosis;18 KsucK, KsucD, FKsuc, LKsuc, Ksuc*D and YKsuc in E.coli BW25113; and KsucE in E.coli DH10B19 as well as those in protozoan parasite, T. gondii, the motifs of which were Ksuc***K, I***Ksuc, LKsuc, QKsuc and KsucG.3 The preference for lysine and arginine residues flanking lysine succinylated sites is a unique feature of lysine succinyltransferase in V. parahaemolyticus. Moreover, to better understand the potential function associated with these identified sequence motifs, we further performed enrichment analysis for succinyl-proteins clusters, of which each has a type of motif in accordance with GO (containing biological process, molecular function and cellular component), the KEGG pathway and the Pfam domain. The results, as shown in Figure 5 and Table S4-S8 in Supporting Information, showed that the succinyl-proteins with different functions, pathways or cellular components have significant preference for adopting different motifs. For example, succinyl-proteins associated with different PEGG pathways have different lysine succinylated motifs. Those proteins that are involved in a pathway of aminoacyl-tRNA biosynthesis preferred to adopt the motif Ksuc******R, while pathways of the biosynthesis of secondary metabolites, microbial metabolism in diverse environments, carbon metabolism, metabolic, glycolysis/gluconeogenesis, pentose phosphate pathway and the citrate cycle tended to adopt the motif K******Ksuc. Overall, the enrichment analysis of succinyl-proteins based on the motifs that they possess clearly illustrates lysine succinylation in V. parahaemolyticus. It provides a better method to understand the potential functions of succinyl-proteins in cells proteome-wide.

Overlap between Lysine Succinylation and Acetylation in V. parahaemolyticus 15



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Currently, the lysine residue has been shown to be a preferred modification site for diverse types of PTMs. Several PTMs, such as methylation, ubiquitylation, SUMOylation, biotinylation, acetylation,15 succinylation, malonylation, glutarylation20 and 2-hydroxyisobutyrylation,32 can occur at this amino acid. In our previous work, we identified lysine acetylation proteome-wide in V. parahaemolyticus.30 Thus, to determine if the succinylation and acetylation occur at the same lysine site, we compared the lysine succinylation data identified in the present study to the previously studied lysine acetylation data30 for the level of modification sites and proteins (Figure 6 and Table S9 in Supporting Information). The comparison results showed that 517 lysine succinylated peptides (26.7% of total succinyl-peptides) matched at 288 proteins (44.9% of total succinyl-proteins), which were acetylated at the same position (Figure 6A). Among the 288 proteins with overlapping modifications, 103 proteins (16% of total succinyl-proteins) had exactly the same succinylation and acetylation lysine sites. Of these 103 proteins, 71 proteins had only one modification site, while 26, 5 and 1 proteins had two, three and four modification sites, respectively (Figure 6B). The other 176 overlapping proteins that had one or more modified lysine residue(s) had at least one different modification lysine site. A representative of overlapping sites between succinylation and acetylation in a protein, Succinyl-CoA ligase beta subunit, is presented in Figure 6C. In this protein, a total of 6 succinylation sites at K56, K66, K70, K75, K146 and K175 were identified (Figure S1 in Supporting Information), while three sites at K66, K75 and K175 were also determined to be acetylated (Figure 6C). This finding of extensive overlap between lysine succinylation and lysine acetylation suggests that succinylation and acetylation frequently occur at the same lysine residues. The observation of significant overlap between these two types of PTMs in proteins has 16


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also been reported in E. coli15 and M. tuberculosis.18 Due to the significant enrichment of succinyl- and acetyl-proteins related to metabolism and protein biosynthesis (in particular ribosome) in V. parahaemolyticus, which was determined in this work and our previous work,30 respectively, we further analyzed the overlap of two protein clusters around the ribosome and central metabolism. As shown in Figure 7A, all of the proteins in the small subunit of the ribosome were found to be modified, and most of the proteins were modified both by succinylation and acetylation. Only two proteins had one type of modification, S16 and S12 modified by succinylation and acetylation alone, respectively. Similarly, besides three proteins (L32, L35 and L36) that were not identified to be succinylated and acetylated, 24 of 31 total modified proteins in the ribosome large subunit were both modified by the two types of modification. The remaining six proteins, L3, L21, L27, L29, L31 and L33, were succinylated alone, while L34 was acetylated alone (Figure 7A). In summary, 82.7% (43/52) of the modified proteins in the ribosome are overlapping, and the percentage is much higher than that of the total overlapped proteins (288) to total modified proteins (1010), i.e., 28.5%. Moreover, another protein-enriched cluster associated with central metabolism was found to be greatly overlapped. Among 38 enzymatic proteins in the glycolysis/gluconeogenesis TCA cycle and pentose phosphate pathway, 33 proteins (86%) were modified both by succinylation and acetylation (Figure 7B and Table S10 in Supporting Information), and the percentage of overlapped proteins was also significantly higher than the ratio of the total overlapped proteins to the total modified proteins (28.5%). Only one protein, the glucose-specific IIBC component of the PTS system (PTSBC), was identified to be succinylated alone, while another four proteins (triosephosphate isomerase (TPI) in glycolysis/gluconeogenesis, iron-sulfur protein of 17



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fumarate reductase (FRB) in TCA cycle, glucose-6-phosphate 1-dehydrogenase (GPDH) and ribulose-phosphate 3-epimerase (RUPE) in pentose phosphate pathway) were found to be modified by acetylation alone. These results, taken together, reflect that the two types of lysine modification are highly enriched and extensively overlapped in proteins related to ribosomes and metabolism, suggesting that both types modification may play important roles in regulating cell processes, especially in central metabolism and ribosomes.

CONCLUSIONS In summary, we report the first global analysis of lysine succinylation in V. parahaemolyticus using a highly sensitive proteomic method. A total of 1931 lysine succinylated sites matched 642 proteins identified in the bacterium, and these succinyl-proteins account for 13.3% of the total proteins in the cell. Bioinformatics analysis showed that these proteins were involved in almost every cellular process in cells, particularly in protein biosynthesis and diverse metabolic pathways. Moreover, the analysis of the sequence motifs demonstrated that the lysine sites flanked by K or R at the -8, -7 or +7 position and without these residues at the -1 or +2 position have a tendency to be succinylated. Notably, the 5 identified motifs differ from those found in other bacteria and eukaryotic cells; however, interestingly, some of these motifs were similar to parts of the acetylation motifs that were also identified in this bacterium. When comparing the succinylation datasets to those for acetylation, 517 succinylated lysines on 288 proteins were found to be acetylated, indicating extensive overlap between these two PTMs. Although the potential regulatory roles and mechanisms for lysine succinylation, also for acetylation, remained to be revealed both in V. parahaemolyticus and other bacteria, our systemic analysis of lysine 18


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succinylation and the overlap between lysine succinylation and acetylation provides a promising start point for the intensive exploration of these two PTMs in V. parahaemolyticus.

ASSOCIATED CONTENT Supporting Information Supplemental figures: Figure S1, Spectra of six identified lysine-succinylated peptides of SucC; Figure S2, Identified enzymatic proteins association with glycolysis/gluconeogenesis, pentose phosphate and citrate cycle; Supplemental tables: Table S1, All identified succinylated-lysine peptides and proteins; Table S2, Functional annotation and subcellular localization of succinyl-proteins; Table S3, Enrichment analysis of GO (biological process, molecular function and cellular component), KEGG and Pfam domain; Table S4-S8, Enrichment analysis of succinylated proteins with motifs 1-5, respectively; Table S9, Overlap analysis of lysine succinylation and acetylation; Table S10, Overlap between succinylation and acetylation of enzymes involved in central carbon metabolism. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * Dr. Jianyi Pan, Institute for Proteomics, School of Life Sciences, Zhejiang Sci-Tech University, 310018, Hangzhou, China. E-mail: [email protected], Tel: +86 571 86843748, Fax: +86 571 86843745. Notes The authors declare no competing financial interest. 19



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ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (31200110), the Natural Science Foundation of Zhejiang Province (LY15C010004), the 521 Talent Program of Zhejiang Sci-Tech University and Zhejiang Provincial Top Key Discipline of Biology.

ABBREVIATIONS PTSA, PTS system, glucose-specific IIA component; PTSBC, PTS system, glucose-specific IIBC component; G6PI, Glucose-6-phosphate isomerase; PFKA, 6-phosphofructokinase; FBP, Fructose-1,6-bisphosphatase class 1; FBA, Fructose-bisphosphate aldolase, class II; TPI, Triosephosphate isomerase; GAPD1, Glyceraldehyde 3-phosphate dehydrogenase; GAPD2, Glyceraldehyde 3-phosphate dehydrogenase; PGK, Phosphoglycerate kinase; PGM, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase; ENO, Enolase; PYK1, Pyruvate kinase; PYK2, Pyruvate kinase; PYD1, Pyruvate dehydrogenase E1 componen; PYD2, Pyruvate dehydrogenase E2 component (dihydrolipoamide acetyltransferase); DLDH, Dihydrolipoyl dehydrogenase; CS, Citrate synthase; AH, Aconitate hydratase 2; ICDH, Isocitrate dehydrogenase; OGD1, 2-oxoglutarate dehydrogenase, E1 component; OGD2, 2-oxoglutarate dehydrogenase E2 component; SUCL, Succinyl-CoA ligase subunit beta; SDHB, Succinate dehydrogenase, iron-sulfur protein; SDHA, Succinate dehydrogenase, flavoprotein; FRA, Fumarate reductase, flavoprotein subunit; FRB, Fumarate reductase, iron-sulfur protein; FRD, Fumarate reductase subunit D; FHI, Fumarate hydratase, class I; MDH, Malate dehydrogenase; PEPCK, Phosphoenolpyruvate carboxykinase; GPDH, 20


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Glucose-6-phosphate 1-dehydrogenase; PGDH, 6-phosphogluconate dehydrogenase; RPIA, Ribose-5-phosphate isomerase A; RUPE, Ribulose-phosphate 3-epimerase; TK1, Transketolase 1; TK2, Transketolase 2; TAL, Transaldolase.

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Tables

Table 1. Comparison of the lysine-succinylated sites and succinyl-proteins of V. parahaemolyticus to those found in other bacterial species. Number of

Number of

Number of

succinylation

succinylated

total

sites a

proteins

proteins

V. parahaemolyticus RIMD 2210633

1931(40)

642

4822

This study

M. tuberculosis H37Rv

1,545(30)

626

4018

17

M. tuberculosis XDR

1739(30)

686

4222

16

E. coli K-12 strain DH10B

2,580(20)

670

4128

15

E. coli K-12 strain BW25113

2,572(45)

990

4,141

13

Species

a

The cut-off score of the peptides is indicated in parentheses.

27


References


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Table 2. Motif analysis of lysine succinylated peptides in V. parahaemolyticus Foreground

Motif No.

Background

Fold

Motif Score

Matches

Size

Matches

Size

Increase

1

*K*******Ksuc*********

6.26

161

1638

845

12775

1.49

2

**K******Ksuc*********

4.76

148

1477

844

11930

1.42

3

**R******Ksuc*********

6.65

142

1780

689

13464

1.56

4

*********Ksuc******K**

4.51

126

1329

728

11086

1.44

5

*********Ksuc******R**

4.14

94

1203

537

10358

1.51

28


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Caption for Figures

Figure 1. Distribution of lysine succinylated peptides based on their length (A) and succinyl-proteins based on the number of succinylated peptides that they contained (B).

Figure

2.

Gene

Ontology

(GO)

functional

annotation

of

the

identified

succinyl-proteins in terms of biological processes (A), molecular functions (B) and subcellular localization (C).

Figure 3. Enrichment analysis of the succinylated proteins in V. parahaemolyticus. (A) GO enrichment for biological processes (blue bars) (P

Global Analysis of Protein Lysine Succinylation Profiles and Their

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